Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Author:

Agam Ganesh,Gebhardt Christian,Popara Milana,Mächtel Rebecca,Folz Julian,Ambrose Benjamin,Chamachi Neharika,Chung Sang Yoon,Craggs Timothy D.ORCID,de Boer Marijn,Grohmann Dina,Ha TaekjipORCID,Hartmann Andreas,Hendrix JelleORCID,Hirschfeld Verena,Hübner Christian G.,Hugel Thorsten,Kammerer Dominik,Kang Hyun-Seo,Kapanidis Achillefs N.ORCID,Krainer GeorgORCID,Kramm Kevin,Lemke Edward,Lerner EitanORCID,Margeat Emmanuel,Martens Kristen,Michaelis JensORCID,Mitra JabaORCID,Moya Muñoz Gustavo G.,Quast Robert,Robb Nicole B.,Sattler MichaelORCID,Schlierf MichaelORCID,Schneider Jonathan,Schröder Tim,Sefer Anna,Tan Piau Siong,Thurn Johann,Tinnefeld Philip,van Noort John,Weiss ShimonORCID,Wendler Nicolas,Zijlstra Niels,Barth Anders,Seidel Claus A. M.,Lamb Don C.,Cordes Thorben

Abstract

AbstractSingle-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than ± 0.06, corresponding to an interdye distance precision of ≤ 0.2 nm and accuracy of ≤ 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to ≲ 10% of the Förster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

Publisher

Cold Spring Harbor Laboratory

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